PROJECT SUMMARY Human cytomegalovirus (hCMV) infects much of the world?s population and establishes a life-long persistent infection defined by both lytic and latent states, and hCMV continues to be a significant issue in human health. hCMV is the leading cause of non-heritable birth defects in the United States and likely around the world. If the virus crosses the placental barrier, hCMV replicates in fetal neural progenitor cells resulting in central nervous system damage. Several antiviral treatments exist but are not approved for use during pregnancy due to toxicity and potential teratogenic effects. The collaborative project described in this application was initiated to address the significant deficiencies in understanding the underlying pathogenesis of congenital hCMV infection and the lack of antiviral approaches. hCMV expresses a viral kinase, pUL97 that regulates diverse viral and cellular processes and can be inhibited by the antiviral compound, maribavir. One target of pUL97 that is important for hCMV replication and maribavir activity is the cellular Tip60 acetyltransferase. Tip60 is a tumor suppressor, manipulated by numerous viruses, and regulates stem cell renewal and differentiation during tissue development. Tip60 is expressed in several isoforms and is part of larger multiprotein Tip60 complexes where specific functional activity is determined by the associated proteins. The objectives of this proposal are twofold: (1) To define the impact of hCMV and the role of the hCMV pUL97 kinase in regulating cellular Tip60 acetyltransferase during neural cell differentiation into functional 3- dimensional tissues, and (2) to assess the hCMV kinase inhibitor maribavir in neural tissue. We hypothesize that hCMV pUL97 manipulates Tip60 to stimulate viral DNA synthesis resulting in alteration of neural tissue development and sensitization of differentiating cells to maribavir anti-hCMV activity. This will be tested in two aims: Aim 1 Test the hypothesis that the contribution of pUL97 to hCMV infection is dependent on tissue-specific expression of Tip60 isoforms during neurogenesis; and Aim 2 Test the hypothesis that pUL97 kinase inhibition prevents hCMV spread in 3-dimensional human neural tissues and allows uninfected human neural progenitor cells to differentiate into a spatial network of astrocytes and synaptically-connected cortical neurons. A comprehensive repertoire of cellular and molecular biological techniques combined with innovative approaches in tissue engineering and mass spectrometry-based imaging will be used to study hCMV infection in developing cortical tissue. Congenital hCMV infection can have devastating effects on children, pose significant challenges for parents, and continues to be a significant burden on society. Regardless of the validity of our hypothesis, these studies will uncover fundamental aspects of hCMV pUL97 kinase biology and Tip60?s contributions to neurogenesis, hCMV infection, and antiviral susceptibility in a clinically relevant cell and tissue type.